左归丸通过调控p38 MAPK/ERK信号通路抑制乳腺癌诱导的破骨细胞活化及机制
Mechanism of Zuoguiwan in Inhibiting Osteoclast Activation Induced by Breast Cancer via Regulating p38 MAPK/ERK Signaling Pathway
付剑江 1梅殷珑 2麻俊超 2朱小翠 2王伟 2吕红2
作者信息
- 1. 江西中医药大学,南昌 330004;恶性肿瘤中医药诊疗与康复江西省重点实验室,南昌 330004
- 2. 江西中医药大学,南昌 330004
- 折叠
摘要
目的:探讨左归丸的抗乳腺癌诱导的破骨细胞活化及机制.方法:为了模拟乳腺癌诱导的破骨性骨转移,该实验采用含50％乳腺癌细胞MDA-MB-231培养上清的条件培养基培养RAW264.7细胞;实验中左归丸的给药浓度分别为5％、10％的左归丸含药血清.抗酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞活化情况;酶联免疫吸附测定法(ELISA)检测RAW264.7细胞Cathepsin K分泌;实时荧光定量聚合酶链式反应(Real-time PCR)检测骨钙素(OCN)、唾液酸蛋白(BSP)mRNA表达水平;采用免疫共沉淀法检测Runt-相关转录因子2(Runx2)与核心结合因子β亚基(CBF-β)间相互作用;蛋白免疫印迹法(Western blot)检测Runx2、磷酸化(p)-Runx2、细胞外调节蛋白激酶1/2(ERK1/2)、p-ERK1/2、p38丝裂原活化蛋白激酶(MAPK)、p-p38 MAPK及CBF-β等蛋白表达.结果:与空白组比较,MDA-MB-231细胞上清组TRAP阳性细胞数目、Cathepsin K分泌显著增加;p-Runx2蛋白表达、与CBF-β相互作用结合能力、BSP和OCN mRNA表达、p-p38 MAPK和p-ERK1/2蛋白表达显著降低(P＜0.01).与MDA-MB-231细胞上清组比较,左归丸含药血清可显著减少TRAP阳性细胞数目、Cathepsin K分泌(P＜0.01),显著增加p-Runx2蛋白表达、BSP和OCN mRNA表达、p-p38 MAPK和p-ERK1/2蛋白表达,并促进Runx2与CBF-β相互作用(P＜0.01),Runx2表达则未见明显改变.与空白组比较,BVD-523组p-p38 MAPK和p-ERK 1/2蛋白表达显著降低(P＜0.01);与BVD-523组比较,低、高浓度左归丸含药血清组p-p38 MAPK表达显著升高(P＜0.01),高浓度左归丸含药血清组p-ERK1/2表达也显著升高(P＜0.01),低剂量组差异无统计学意义.结论:左归丸可以通过诱导破骨细胞内骨形成的关键性转录调节蛋白Runx2磷酸化而抑制破骨细胞活化,这一过程与p38 MAPK/ERK信号转导途径的活化密切相关.
Abstract
Objective:To investigate the effects of Zuoguiwan on osteoclast activation induced by breast cancer and its mechanism.Methods:To simulate breast cancer-induced osteoclastic bone metastasis,RAW264.7 cells were cultured in conditioned medium containing 50％supernatant of MDA-MB-231 breast cancer cells.The dosages of Zuoguiwan used in the experiment were sera containing 5％and 10％Zuoguiwan.Tartrate-resistant acid phosphatase(TRAP)staining was used to detect osteoclast activation.Enzyme-linked immunosorbent assay(ELISA)was used to measure Cathepsin K secretion from RAW264.7 cells.Real-time quantitative polymerase chain reaction(PCR)was used to detect the mRNA expression levels of osteocalcin(OCN)and bone sialoprotein(BSP).Immunoprecipitation was employed to detect the interaction between Runt-related transcription factor 2(Runx2)and core binding factor β subunit(CBF-β).Western blot was used to assess the protein expression of Runx2,phosphorylated Runx2(p-Runx2),extracellular signal-regulated kinases 1/2(ERK1/2),p-ERK1/2,p38 mitogen-activated protein kinase(MAPK),p-p38 MAPK,and CBF-β.Results:Compared with the blank group,the MDA-MB-231 cell supernatant group showed a significant increase in TRAP-positive cell counts and Cathepsin K secretion.Meanwhile,the expression levels of p-Runx2,Runx2-CBF-β interaction,BSP and OCN mRNA,p-p38 MAPK,and p-ERK1/2 proteins were significantly decreased(P＜0.01).Compared with the MDA-MB-231 cell supernatant group,Zuoguiwan-containing sera significantly reduced TRAP-positive cell counts and Cathepsin K secretion(P＜0.01),significantly increased p-Runx2,BSP and OCN mRNA expression,as well as p-p38 MAPK and p-ERK1/2 protein levels,and promoted the interaction between Runx2 and CBF-β(P＜0.01).No significant change in Runx2 expression was observed.Compared to the blank group,the BVD-523 group showed significantly lower expression of p-p38 MAPK and p-ERK1/2 proteins(P＜0.01).Compared with the BVD-523 group,both low and high concentration Zuoguiwan-containing sera groups showed significantly higher p-p38 MAPK expression(P＜0.01),and the high concentration Zuoguiwan group also exhibited a significant increase in p-ERK1/2 expression(P＜0.01),while no statistical difference was found in the low-dose group.Conclusion:Zuoguiwan inhibits osteoclast activation by inducing phosphorylation of the key transcriptional regulator Runx2 in intra-osteoclast bone formation,and this process is closely associated with the activation of the p38 MAPK/ERK signaling pathway.
关键词
左归丸/破骨性骨转移/乳腺癌/Runt相关转录因子2/p38丝裂原活化蛋白激酶/细胞外调节蛋白激酶/信号通路Key words
Zuoguiwan/osteolytic metastasis/breast cancer/Runt-related transcription factor 2/p38 mitogen-activated protein kinase/extracellular regulated protein kinase 1/2/signaling pathway引用本文复制引用
出版年
2025
国家科技期刊平台
NSTL
维普
万方数据